血管瘤干细胞联合雌激素构建血管瘤动物模型及其机制的研究

Infantile Hemangioma(IH) is the most common benign tumor in children with a unique characteristic course of rapid growth in the first year of life,the incidence of IH has been estimated to be 10% . But its mechanism remains unclear. A perfect animal model would allow new insights into the biological processes involved in the development and pathogenesis of IH..We and other scholars have confirmed that there are positive CD133 stem cells existed in the hemangioma tissue, these cells can form IH liked tissue that expressed specific marker of Glucose transporter type 1(Glut-1) after they were subcutaneous injected into immunodeficiency mice.But the growth of these tissues still could not catch that of the real hemangiomas for a possible cause of different enviroment between patient's body and mice.Considering the estrogen levels are significantly high in patients with hemangiomas in proliferating phase. And its promoted role in the proliferation of hemangiomas,combined with the new discovery of the change of peroxisome proliferatoractivated receptor-γ(PPAR-γ), which is important in cell differentiation and apoptosis in hemangioma,we hypothesize estrogen can prevent hemangioma derived stem cells differentiating into adipocytes and apoptosis through disintegrating PPAR-γ, promoting the proliferation.Base on this hypothesis,this research was designed to study the influence of estrogen to hemangioma derived stem cells,including differentiation ability,apoptosis and proliferation,as well as change in the pathway of PPAR-γ target gene,protein. On the other hand,estrogen antagonists will be administered in the control group to confirm this effect.For the first time,combine with the study of a possible effect of enhancing self-renew and tumorigenic capability of HemSC by 3D-culture,a mimic hemangioma mice model will be established by blending subcutaneous injection of hemangioma derived stem cells,human umbilical vein endothelial cells and estrogen which can provide a platform for the research of the mechanism and evaluation of treatment.

血管瘤是婴幼儿最常见的良性血管肿瘤，1岁以下儿童发病率约为10%。因其发病机制不清，缺乏可用的实验模型，影响了对其发病机制和治疗方法的研究。近期我们及国外学者均证实，血管瘤组织中存在CD133阳性的干细胞，注入裸鼠皮下后，能够形成表达血管瘤特异标记Glut-1的血管组织，但其增殖较慢，与真正的血管瘤尚有差距。根据已有的雌激素在增殖期血管瘤中表达增高且能促进血管瘤生长的特性，结合最近发现的在细胞分化和凋亡过程中发挥重要作用的PPAR-γ核转录因子的变化，假设雌激素通过下调PPAR-γ而抑制血管瘤干细胞向脂肪细胞分化和凋亡，保持多能性，促进其增殖，由此研究雌激素在体外对血管瘤干细胞分化、凋亡、增殖的影响，PPAR-γ及其靶基因、蛋白质的变化，利用雌激素拮抗剂验证其作用，并研究三维培养促进血管瘤干细胞自我更新和成瘤性的作用，将血管瘤干细胞和脐静脉内皮细胞混合，结合雌激素，构建新型可用的动物模型。

血管瘤是婴幼儿最常见的先天性良性血管肿瘤。目前影响血管瘤治疗效果进一步提高的主要原因是对血管瘤的病因和发病机制了解不够以及缺乏理想的血管瘤动物模型。有研究表明血管瘤干细胞注入裸鼠皮下能形成表达血管瘤特异标记Glut-1的血管组织，但是并没有表现出血管瘤组织在增殖期快速增长的特性，这可能与细胞微环境有关。根据已有的雌激素在增殖期血管瘤中表达增高且能促进血管瘤生长的特性，本项目利用婴幼儿血管瘤（IH）干细胞（HEMSC）联合脐静脉内皮细胞（HUVEC）结合肌注雌激素,探索构建血管瘤裸鼠模型的新方法，研究雌激素（17β-雌二醇，E2）对血管瘤的作用机制，探讨Notch信号通路在血管瘤发生、发展过程中的调控作用。对体外培养的HEMSC给予E2刺激，研究E2对HEMSC的增殖以及对FGF2、VEGF-A、雌激素受体（estrogen receptor ERα）、Notch基因家族受体Notch1及配体Jagged1影响，来探讨E2的作用机制。将HEMSC与脐静脉内皮细胞（HUVEC）联合注入裸鼠皮下，测定成瘤能力。研究发现HemSC与HUVEC联合注射并结合肌注E2，能够形成更多的微血管。随着E2浓度的不断升高，MVD也逐渐增大，而且血管瘤内皮细胞向脂肪细胞转化的时间也相应延长。体外实验发现，当E2浓度为10-9M～10-6M时，能够明显促进HEMSC增殖。高于10-6M后,HEMSC的增殖变得不明显。E2能明显促进HEMSC表达VEGF-A、FGF2 mRNA和蛋白，基因与蛋白的表达与浓度梯度呈线性关系。免疫组织化学染色示，ERα及葡萄糖转运蛋白1（Glut-1）在IH组织中高表达。Notch基因家族Notch1及配体Jagged1，随E2浓度的增高而表达增强。综上所述，将HEMSC和HUVEC混合注入裸鼠皮下结合肌注 E2，能够成功构建血管瘤模型，明显增加瘤体MVD,并且能更好的模仿血管瘤的自然病程特点。E2能够有效促进HemSC增殖，并抑制其向脂肪细胞转化，在HemSC分化为血管内皮细胞、周细胞的过程中起到了重要的调控作用。ERα对Notch 信号通路中受体 Notch1及配体Jagged1具有调控作用，雌激素受体可能通过对 Notch 信号通路的调控，来影响HemSC分化为其他细胞的能力。雌激素促血管生成的作用主要通过上调VEGF-A、FGF2的表达使两者协同而实现。